Selective and potent inhibitory effect of docosahexaenoic acid (DHA) on U46619-induced contraction in rat aorta

Abstract

Inhibitory effects of docosahexaenoic acid (DHA) on blood vessel contractions induced by various constrictor stimulants were investigated in the rat thoracic aorta. The inhibitory effects of DHA were also compared with those of eicosapentaenoic acid (EPA) and linoleic acid (LA). DHA exhibited a strong inhibitory effect on the sustained contractions induced by U46619, a TXA(2) mimetic. This inhibitory effect of DHA was not affected by removal of the endothelium or by treatment with either indomethacin or N(ω)-nitro-l-arginine. DHA also significantly diminished PGF(2α)-induced contraction but did not show any appreciable inhibitory effects on the contractions to both phenylephrine (PE) and high-KCl. Similarly, EPA exhibited significant inhibitory effects against the contractions induced by both U46619 and PGF(2α) without substantially affecting either PE- or high-KCl-induced contractions. However, both DHA and EPA generated more potent inhibitions against contractions induced by U46619 than those by PGF(2α). In contrast, LA did not show significant inhibitory effects against any contractions, including those induced by U46619. The present findings suggest that DHA and EPA elicit more selective inhibition against blood vessel contractions that are mediated through stimulation of prostanoid receptors than those through α-adrenoceptor stimulation or membrane depolarization. Although DHA and EPA have similar inhibitory potencies against prostanoid receptor-mediated contractions, they had a more potent inhibition against TXA(2) receptor (TP receptor)-mediated contractions than against PGF(2α) receptor (FP receptor)-mediated responses. Selective inhibition by either DHA or EPA of prostanoid receptor-mediated blood vessel contractions may partly underlie the mechanisms by which these ω-3 polyunsaturated fatty acids exert their circulatory-protective effects.

Fig. 1.

Inhibitory effects of DHA on the sustained contraction to U46619 in segments of the rat thoracic aorta. A–C: Typical traces showing the inhibitory effects of DHA vs. U46619. Firstly, acetylcholine (ACh, 10^–5 M) was applied during the contraction to noradrenaline (NA, 3 × 10^–7 M). The preparations resulting in traces A and C were judged to be endothelium-intact (+EC) since ACh produced an almost full relaxation against the NA-induced contraction. The preparation used for trace B was verified as endothelium-denuded (-EC) by the disappearance of ACh-induced relaxation. Inhibitors were not present in A and B whereas both indomethacin (Indo, 3 × 10^–6 M) and N^ω-nitro-l-arginine (l-NNA, 5 × 10^–5 M) were present in C. w: wash of preparation with fresh medium. D: Summarized data showing the inhibitory effects of DHA vs. U46619. Data are shown as mean values ± S.E.M. (n = 4 for each). No significant differences were found among the three groups.

Fig. 2.

A typical trace showing the effects of DHA on NA-induced sustained contractions in preparations of the rat aorta with intact endothelium. The preparation used was endothelium-intact since an almost full relaxation was produced by ACh (10^–5 M) during the sustained contraction to NA (3 × 10^–7 M). Sustained contraction was again induced by NA (3 × 10^–7 M), and DHA (10^–5 M) was applied to the bath medium. To examine whether DHA exerted any effects on NA-induced contraction, tension changes were recorded for 60 min after DHA application. To verify the functional integrity of endothelium, appearance of relaxant response to ACh (10^–5 M) was confirmed at the end of experiments. Tension changes were recorded in the absence of Indo and l-NNA. Similar experiments were performed in total n = 4 preparations. w: wash of preparation with fresh medium.

Fig. 3.

Effects of DHA on the sustained contractions to various stimulants. A–D: Typical traces showing the effects of DHA (10^–5 M) on the sustained contractions to U46619 (5 × 10^–9 M) (A), PGF2α (10^–5 M) (B), phenylephrine (PE, 3 × 10^–7 M) (C) and high-KCl (8 × 10^–2 M) (D). Preparations used were endothelium-denuded, and tension changes were recorded in the presence of Indo (3 × 10^–6 M). PPV: papaverine, 10–4 M; w: wash of preparation with fresh medium. E: Summarized data showing the inhibitory effects of DHA including those of DHA vs. noradrenaline (NA, 10^–7 M). F: Developed tensions (contraction) attained with tested constrictor stimulations. Muscle tensions were shown being normalized with respect to high-KCl (8 × 10^–2 M)-induced muscle tension obtained in the beginning of experiments. Statistical significances were detected between: U46619 vs. PE, NA and high-KCl, ^a)P < 0.01; PGF2_α vs. PE, NA and high-KCl, ^b)P < 0.01; PE vs. high-KCl ^c)P < 0.05.

Fig. 4.

Pretreatment effects of DHA on the aortic contractions to various stimulants. A, B: Typical traces showing the pretreatment effects of DHA (10^–5 M) (B) or its vehicle (A) vs. U46619 (10^–8 M). Tension changes were recorded in the presence of Indo (3 × 10^–6 M) with endothelium-denuded preparations. w: wash of preparation with fresh medium. C–H: Summarized data showing the pretreatment effects of DHA on various contractions. Contractile stimulants used were: U46619 (10–8 M) (C), PGF_2α (10^–5 M) (D), PE (3 × 10^–7 M) (E), NA (10^–7 M) (F), 5-hydroxytryptamine (5-HT, 10^–5 M) (G) and high-KCl (8 × 10^–2 M) (H). Data are shown as mean values ± S.E.M. (n = 4 for each). Significant differences between two groups: ^a)P < 0.01, ^b)P < 0.05.

Fig. 5.

Comparison of the inhibitory effects of DHA, EPA and LA on the sustained contractions induced by various stimulants. Preparations used were endothelium-denuded, and tension changes were recorded in the presence of Indo (3 × 10^–6 M). Sustained contractile stimulants were: U46619 (5 × 10^–9 M) (A), PGF_2α (10^–5 M) (B), PE (3 × 10^–7 M) (C) and high-KCl (8 × 10^–2 M) (D). When the muscle tension increased with vasoconstrictor stimulations reached a steady-state level, each PUFA (DHA, EPA, LA) at a desired concentration was applied to the bath medium. The PUFA concentrations were 10^–6 – 3 × 10^–5 M. The data with 10^–5 M DHA vs. U46619, PGF_2α, PE and high-KCl are the same shown in Fig. 3. Tension developments attained with tested constrictor stimulants are also shown in E. Data are shown as mean values ± S.E.M. (n = 4 for each in A–D, and n = 16 for each in E). Statistical significances were detected between: U46619 vs. PE and high-KCl, ^a)P < 0.01; PGF2α vs. PE and high-KCl, ^b)P < 0.01.